This invention relates to the application of nuclear magnetic resonance (NMR) imaging (tomography) to simultaneous, non-destructive chemical and structural analysis of an object or sample while it is subjected to rotational or other periodic motion. Although not so limited, the invention has particular utility in the analysis of solid objects. NMR spectroscopy can provide great specificity in chemical analysis, in addition to information on molecular motions and physical states. A combination of NMR spectroscopy with imaging in solid samples makes possible a wide variety of applications in industry, medicine and biology, such as analysis of fabricated ceramic or polymeric parts, and imaging of phosphorus in bone. The method of this invention as applied to a rotating liquid sample makes possible the improved separation and analysis of protein mixtures.
The use of NMR in chemical analysis of liquid samples is a well established art. E. D. Becker, "High Resolution NMR, " 2nd edition, Academic Press, New York, 1980, provides a good review of the subject and of numerous variations in NMR techniques for liquid samples. ln general, the liquid sample is placed in the static magnetic field of the instrument. All magnetic nuclei (those possessing spin angular momentum--there is at least one such isotope for every chemical element) of the sample will display a tendency to align with the field. One or more radiofrequency (RF) pulses applied to the sample via the inductance of a tuned electrical circuit will cause the net magnetization of one such isotope to be nutated to a direction transverse to the field. This transverse magnetization will precess about the field direction at a nominal frequency (the Larmor frequency) characteristic of the isotope and proportional to the strength of the field. (The radiofrequency must be near the Larmor frequency for the RF pulses to have substantial effect.) For example protons (the most common nuclear isotope of hydrogen) will precess at about 42.57 MHz in a field of 1.0 Tesla (10,000 gauss). Chemical information is obtained primarily by measuring the small frequency shifts in Larmor frequency which are caused by differences in the shielding of the nuclei from the field by the molecular environment of nuclei. This effect is called the chemical shielding or chemical shift, and is typically displayed as a frequency spectrum of intensity of signal versus frequency or chemical shift. Thus, with respect to a certain reference compound, e.g., tetramethylsilane, protons in a methyl group are typically shifted about one part per million (ppm) in frequency downfield (equivalent to higher frequency), while methylene protons are shifted about two ppm downfield. The detection and measurement process is actually comprised of detecting the RF voltage induced in the tuned circuit by the precessing magnetization as it decays to its unperturbed state (the "free induction decay" or FID), digitizing the FID signal, and performing a Fourier transform operation on the signal with a computer to obtain the frequency spectrum.
The term "spectrosocopy" will be used to imply measuring frequency spectra with primary emphasis on such chemical information, as distinct from the term "imaging", which will refer primarily to spatial information content. An FID signal may contain both spectroscopic and imaging information.
Analysis of solid samples by NMR spectroscopy is through analogous means, although complications in the physics of nuclear magnetic moments in solids creates unusual demands on the instruments and requires special NMR techniques. For this reason, solid state NMR is not nearly as widely applied as is liquid state NMR. Reviews of NMR spectroscopy of solids include:
M. Mehring, "High Resolution NMR Spectrosocopy in Solids," number 11 in "NMR--Basic Principles and Progress," Springer-Verlag, Berlin, 1976; also 2nd edition, 1983.
U. H. Haeberlen, "High Resolution NMR in Solids: Selective Averaging," supplement 1 in "Advances in Magnetic Resonance," Academic Press, N.Y., 1976.
Some specific NMR methods for solids are disclosed in:
U.S. Pat. No. 3,474,329, issued Oct. 21, 1969 to J. S. Waugh. PA1 U.S. Pat. No. 3,530,373, issued Sept. 22, 1970 to J. S. Waugh. PA1 U.S. Pat. No. 3,530,374, issued Sept. 22, 1970 to U. H. Haeberlen & J. S. Waugh. PA1 U.S. Pat. No. 3,792,346, issued Feb. 12, 1974 to M. G. Gibby et al. PA1 P. Mansfield and P. K. Grannell, "Diffraction" and Microscopy in Solids and Liquids by NMR, Physical Review B, Vol. 12, No. 9, 3688-3634 (1975). PA1 R. A. Wind and C. S. Yannoni, Selective Spin Imaging in Solids, Journal of Magnetic Resonance 36, 269-272 (1979). PA1 U.S. Pat. No. 4,301,410, issued Nov. 17, 1981 to R. A. Wind and C. S. Yannoni. PA1 N. M. Szeverenyi and G. E. Maciel, NMR Spin Imaging of Magnetically Dilute Nuclei in Solid State NMR, Journal of Magnetic Resonance 60, 460-466 (1984). PA1 W. P. Rothwell, D. R. Holecek and J. A. Kershaw, NMR Imaging: Study of Fluid Absorption by Polymer Composites, Journal of Polymer Science: Polymer Letters Edition 22, 241-247 (1984). PA1 A. N. Garroway, J. Baum, M. G. Munowitz and A. Pines, NMR Imaging in Solids by Multiple-Quantum Resonance, Journal of Magnetic Resonance 60, 337-341 (1984).
One such complication in solid state NMR is the fact that chemical shielding is anisotropic; it varies with the orientation of the molecule in the magnetic field. Thus, in a polycrystalline solid containing all orientations, a single chemical group displays a wide range of shielding values. (The rapid, random molecular motion in liquids causes an averaging of these values to a single value, and in fact makes possible the simplicity of liquid NMR.) The anisotropy of many such nuclear interactions may be, in effect, removed with the technique of "magic angle spinning" in which the sample is rapidly rotated (at a rate on the order of 1 to 4 kHz) about an axis inclined to the magnetic field direction at an angle of 54.7.degree. (the "magic" angle). This produces an averaging process which is mathematically equivalent to the averaging caused by moleclar motion. The magic angle technique for averaging of direct spin-spin (direct dipolar) interactions in solids was disclosed by I. J. Lowe in Free Induction Decays of Rotating Solids, Physical Review Letters 2, 285-287 (1959), and for quadrupolar interactions by Andrew, Bradbury and Eades, Archives of Science. Geneva 11, 223 (1958).
The above-mentioned U.S. Pat. No. 3,474,329 discloses NMR apparatus which is programmed to produce RF excitation energy the amplitude and phase of which are such that the effects of spin-spin interactions are averaged to a reduced value. Resonance shifts, although also affected, can still be observed in the output signal. It is stated that the method of operation of such apparatus is useful for solids whose dipolar interactions are large. The method comprises adapting a coherent RF modulator in a pulsed NMR apparatus to provide a particular sequence of phase modulated pulses, or to provide a phase and amplitude modulated continuous wave RF excitation.
The above-mentioned U.S. Pat. Nos. 3,530,373 and 3,530,374 disclose NMR apparatus and a method of operation thereof stated to be useful for solids whose resonance shifts and electron coupled spin-spin interactions are smaller than would otherwise be obscured by static nuclear magnetic dipole-dipole interactions and/or quadrupolar interactions. The method of these patents involves adapting a coherent RF modulator in a pulsed NMR apparatus to provide a particular sequence of phase modulated RF pulses, or a phase modulated RF pulse in conjunction with a video pulse.
U.S. Pat. No. 3,792,346 discloses a method for detecting nuclear magnetic and/or electric quadrupole resonance frequencies of isotopically rare or chemically dilute nuclei in the presence of one or more abundant nuclear spin species in solid samples. The free induction decay (FID) of the dilute nuclei is directly detected after an applied RF field at the Larmor frequency of the dilute nuclei is removed. A high resolution FID of the dilute nuclei is obtained by applying an RF field at the Larmor frequency of the abundant spin system during the detection interval.
In Nature, 242, 190 (1973), P. Lauterbur disclosed the detection of spatial distributions of spin densities and/or relaxation times using high-resolution NMR procedures in combination with magnetic field gradients for localized liquid regions in biological systems. This has led to widespread use of NMR tomography in diagnostic radiology and similar medical applications, all relating to liquid-like materials.
lt should be noted that, for the purposes of NMR analysis, a "liquid" sample is one in which the anisotropy of interactions such as the chemical shift has been substantially averaged by molecular motion. In this context, most components of living tissues are liquid, irrespective of the gross physical characteristics of the tissues. Other components, most notably the mineral phase of bone and teeth, but also certain rigid structural proteins and cell membranes are "solid" in the NMR context.
Another method for producing NMR images of liquid-like components of an object was disclosed by A. Kumar, D. Welti and R. R. Ernst in "NMR Fourier Zeugmatography", Journal of Magnetic Resonance 18, 69-83 (1975). In this method, magnetic field gradient pulses are applied to the object before and during acquisition by computer of the NMR FID signals from the object. In successive acquisitions of the FID signals, some of the field gradients are sequentially incremented in intensity or time duration. Reconstruction of the image requires use of the Fourier transform operation.
As in all methods of NMR imaging, these methods produce encoding of spatial information in the FID through the use of spatially varying magnetic fields (for instance linear gradients of the static magnetic field). Lauterbur's method is generally known as "projection reconstruction" or "zeugmatography," while Kumar, Welti and Ernst's method is generally known as "Fourier imaging". Many NMR imaging methods in current use contain elements of both methods.
NMR imaging of solids has received little attention in comparison to the studies devoted to NMR procedures for medical applications. Other publications relating to NMR imaging of solids of which applicant is aware include the following:
The Mansfield and Grannell article contains various equations, calculations derived therefrom and experimental results in ordered and disordered systems, using either multiple-pulse line-narrowing sequences or single pulses, together with an applied linear magnetic field gradient.
In solid samples where multiple pulse line narrowing techniques are required, it is concluded that a spatial resolution limit of 4 .mu.m in a 100.mu.m thick sample is achievable, or about 25 pixels (picture elements) in a single dimension. The resolution limit is determined by the linewidth achievable by the multiple pulse sequence, the efficiency of which degrades as the intensity of the magnetic field gradient is increased in an attempt to improve the spatial resolution.
The article by Wind and Yannoni reports selective spin imaging in solids. It is pointed out that in solids the problem of resolution is difficult to overcome because the natural linewidth usually caused by static dipolar interactions is about 10 to 50 kHz. For a resolution of 0.5 mm this would require gradients varying from 0.2 to 1 MHz/cm which are difficult to obtain, especially for larger objects. The solution to the problem resides in the application of line-narrowing techniques wherein the dipolar broadening can be reduced by a factor of 100 to 400. The method applied by the authors involved a combination of RF irradiation and field modulation for line-narrowing. Line narrowing was obtained for specific values of the RF amplitude, modulation frequency and index, and the offset from resonance. A train of identical RF pulses with equal spacings was used, and the field modulation was replaced by frequency modulation. It was found that line-narrowing could be obtained for many values of the different parameters. The method of line narrowing used here is rather specific to the nucleus and compound studied. More strongly dipolar coupled solids would not narrow as well. The method is very time-consuming, since data for only one position along a field gradient are acquired in any FID.
The above mentioned U.S. Pat. No. 4,301,410 discloses a method of spin imaging in solids using NMR, wherein a solid sample within the field of an RF excitation coil and the static external magnetic field of an NMR spectrometer is rotated about an axis which makes an angle of 54.7.degree. with the direction of the static external magnetic field. A specific magnetic field gradient is superimposed in one direction on the static external magnetic field in order to provide different resonant NMR frequencies in different parts of the sample, and the magnetic field gradient is rotated synchronously with the sample. Solid state NMR line narrowing procedures must be applied while collecting data. The phase relation is changed between the sample rotation and the field gradient rotation on a step-by-step basis, with data being collected each time, and the spin image of the solid sample is then reconstructed.
The NMR line narrowing procedures are stated to be those disclosed in the above-mentioned U.S. Pat. Nos. 3,530,373; 3,530,374; 3,474,329 and 3,792,346. In addition, the step of spinning the sample around an axis of 54.7.degree. with the static external magnetic field (the so-called "magic angle") is also stated to remove other broadening effects not removed by the procedures disclosed in these four U.S. patents.
The method of U.S. Pat. No. 4,301,410 thus involves magic angle spinning (MAS) with a synchronously rotating magnetic field gradient, during which data are collected while performing solid state NMR line narrowing procedures, in a variety of different phase relations between the sample rotation and the field gradient rotation. This patent alleges that the method is advantageous in extending spin imaging in solids by NMR to a wider class of materials, although no specific examples or other data are disclosed.
The Szeverenyi and Maciel article discloses a method of NMR imaging of carbon in solids. Some line narrowing is achieved by proton decoupling. The samples chosen for study all have unusually narrow carbon NMR lines. This method would be difficult to use or impractical in the general case. It also does not allow resolution of chemical shift information.
The article by Rothwell, et al., describes NMR imaging of a polymer composite sample. Although the sample is solid in outward appearance, these authors have not in fact imaged the solid, but rather liquid water which has penetrated the solid.
The article by Garroway, et al., employs multiple quantum coherence to magnify the effect of a magnetic field gradient to an extent which is sufficiently intense that it overcomes the natural linewidth of a dipolar coupled solid. This method can only work with homonuclear dipole coupled spin systems (or the equivalent quadrupolar coupled spins). It was demonstrated for one sample which has properties convenient for the experiment, and may be very difficult to carry out for a general substance. It is also not capable of chemical shift resolution in more complicated substances.
The present state of the art, with respect to imaging of solid objects by NMR procedures, has not yet made it possible to obtain high spatial resolution imaging of solid objects nor to obtain a chemical image of solid objects. Insofar as other types of imaging are concerned using X-rays, radioisotopes, or ultrasound, it is not possible to derive specific information on chemical composition throughout the object.
There are several methods of obtaining "chemical images" in liquids, i.e., images of specific chemical components as determined by their chemical shift spectra. The one most closely related to this invention is disclosed in U.S. Pat. No. 4,319 190, issued Mar. 9, 1982 to Truman R. Brown. In this method, spatial information is encoded in the FID using magnetic field gradient pulses as in Fourier imaging. All magnetic field gradients are off during acquisition of the FID, which therefore evolves during the acquisition period only according to the chemical shift spectrum. Fourier transformation over each spatial dimension encoded and over the chemical shift dimension produces the chemical shift spectrum for each pixel (picture element) in the spatial image. As disclosed, this method will not in general work for solids, because it does not deal with the broad lines characteristic of solids.
With respect to the use of an ultracentrifuge in combination with optical detection, wherein light is directed through a quartz sample cell as it passes periodically between the source and detector in the ultracentrifuge, there is a need for detection in solutions which are not sufficiently transparent to the wavelength of light used in optical detection, and for detection where refractive index or absorption coefficient variations are too small to be distinguished. In addition, chemical composition which can be obtained, e.g. by ultraviolet wavelengths, is relatively nonspecific.